The present invention relates generally to methods for detecting membrane derived caspase activity and modulators thereof, and more particularly to novel cell-free screening assays for identifying inhibitors and enhancers of membrane derived caspase activity.
Tissue homeostasis is maintained by the process of apoptosisxe2x80x94that is, the normal physiological process of programmed cell death. Changes to the apoptotic pathway that prevent or delay normal cell turnover are often as important in the pathogenesis of diseases as are abnormalities in the regulation of the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is similarly regulated under normal circumstances by the interaction of gene products that either function to prevent or induce cell death.
Since apoptosis functions in maintaining tissue homeostasis in a range of physiological processes, such as embryonic development, immune cell regulation and normal cellular turnover, the dysfunction or loss of regulated apoptosis can lead to a variety of pathological disease states. For example, the loss of apoptosis can lead to the accumulation of self-reactive lymphocytes associated with many autoimmune diseases. Inappropriate loss or inhibition of apoptosis can also lead to the accumulation of virally infected cells and hyperproliferative cells, such as neoplastic or tumor cells. Similarly, the inappropriate activation of apoptosis can contribute to a variety of pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury.
Although apoptosis is mediated by diverse signals and complex interactions of cellular gene products, the results of these interactions ultimately feed into a cell death pathway that is evolutionarily conserved between humans and invertebrates. The pathway, itself is a cascade of proteolytic events analogous to that of the blood coagulation cascade.
Several gene families and products that modulate the apoptotic process have now been identified. One family is the aspartate-specific cysteine proteases (xe2x80x9ccaspasesxe2x80x9d). The caspase Ced-3, identified in C. elegans, is required for programmed cell death during development of the roundworm C. elegans. Ced-3 homologues as well as other caspases have been characterized. The human caspase family includes, for example, Ced-3, human ICE (interleukin-1-xcex2 converting enzyme) (caspase-1), ICH-1 (caspase-2), CPP32 (caspase-3), ICErelII (caspase-4), ICErelIII (caspase-5), Mch2 (caspase-6), ICE-LAP3 (casepase-7), Mch5 (caspase-8), ICE-LAP6 (caspase-9), Mch4 (caspase-10), caspase-11, caspase-12, caspase-13, caspase-14, and others.
The caspase family of cysteine proteases are essential effectors of the apoptotic process (Yuan et al., Cell 75:641-652, 1993; Alnemri et al., Cell 87:171, 1996; Cohen, Biochem. 326:1-16, 1997; Miller, Semin. Immunol 9:35-49, 1997; Salvesen and Dixit, Cell 91:443-446, 1997). Caspases are synthesized as inactive zymogens, which are activated by proteolytic processing to yield large (xcx9c18 kDa) and small (xcx9c12 kDa) subunits that associate to form active enzymes (Thornberry et al., Nature 396:768-774, 1992; Nicholson et al., Nature 376:37-43, 1995; Stennicke and Salvesen, J. Biol. Chem. 272:25719-25723, 1997). Diverse apoptotic stimuli cause the activation of specific caspases which then initiate a protease cascade by proteolytically processing additional caspases (Srinivasula et al., Proc. Natl. Acad. Sci. USA 93:14486-14491, 1996; Yu et al., Cancer Res. 58:402-408, 1998). Once activated, these downstream (executioner) caspases kill cells by cleaving specific molecular targets that are essential for cell viability or by activating pro-apoptotic factors (Liu et al., Cell 89:175-184, 1997; Enari et al., Nature 391:43-50, 1998; Salvesen and Dixit, Cell 91:443-446, 1997). Although caspases have been generally shown to be cytosolic proteins (Miller et al., J. Biol. Chem. 268:18062-18069, 1993; Nicholson et al., Nature 376:37-43, 1995; Li et al., J. Biol. Chem. 272:30299-30305, 1997), immunochemical studies have suggested that in some instances, caspases might also be associated with the nucleus or plasma membrane (Singer et al., J. Exp. Med. 182:1447-1459, 1995; Krajewski et al., Blood 89:3817-3825, 1997; Posmantur et al., J. Neurochem. 68:2328-2337, 1997). Recently published data has also indicated an association of certain caspases with mitochondria and endoplasmic reticulum (Mancini et al., J. Cell Biol. 140:1485-1495, 1998; Chandler et al., J. Biol. Chem. 273:10815-10818, 1998).
The Bcl-2 family constitutes another key set of regulators of the apoptotic pathway. These proteins can function to modulate apoptosis in a wide variety of cell systems (Oltvai and Korsmeyer, Cell 79:189-192, 1994; Reed, Nature 387:773-776, 1997). Bcl-2 family proteins contain one to four conserved domains, designated BH1-BH4, and most family members contain a carboxyl-terminal transmembrane anchor sequence which allows them to be associated with cellular membranes including the outer membrane of the mitochondria, the nuclear envelope and the endoplasmic reticulum (Reed, Nature 387:773-776, 1997; Krajewski et al., Cancer Res. 53:4701-4714, 1993; Yang et al., J. Cell. Biol. 128:1173-1184, 1995; Lithgow et al., Cell Growth Differ 3:411-417, 1994). The over-expression of Bcl-2 has been shown to inhibit the activation of cytoplasmic caspases following apoptoic stimuli in several cell systems (Armstrong et al., J. Biol. Chem. 271:16850-16855, 1996; Chinnaiyan et al., J. Biol. Chem. 271:4573-4576, 1996; Boulakia et al., Oncogene 12:29-36, 1996; Srinivasan et al., J. Neurosci. 16:5654-60, 1996). Moreover, previous work has demonstrated that Bcl-2 inhibits the onset of apoptosis, but once apoptosis is initiated, Bcl-2 does not impede the process (McCarthy et al., J. Cell Biol. 136:215-217, 1997). However, it remains unclear how the membrane bound Bcl-2 exerts control over the soluble cytoplasmic caspases. Further, no suitable methods exist for studying membrane bound Bcl-2 and its effects on caspase activity in a cell free manner.
The identification of compounds that modulate the apoptotic pathway via enhancement or inhibition of membrane derived caspase activity has been hindered by the lack of such methods. Available methods are limited by the lack of specificity, efficiency, and/or utilization of whole cells or cytoplasmic extracts thereof. For example, most anti-cancer drugs are screened for their ability to kill cells and therefore will identify compounds that induce both necrosis or apoptosis. In addition, many other assay techniques focus on studying the inhibition or enhancement of caspase enzymes located further into the cascade. Therefore, there exists a need in the art for methods of identifying compounds that not only inhibit or enhance cell death, but also compounds that modulate the initiation of the apoptotic cascade. The present invention fulfills this need, while further providing other related advantages.
The foregoing characteristics, and others which shall be described in greater detail below, make the methodologies described herein particularly attractive for drug discovery applications.
The present invention generally provides methods for detecting membrane derived caspase activity and methods for identifying modulators thereof. In one aspect, the invention provides a method for identifying membrane derived caspase activity, that includes, incubating a membrane fraction comprising heavy or nuclear membranes under conditions and for a time sufficient to allow for the evolution of caspase activity, and subsequently detecting caspase activity.
In another aspect, the present invention provides a method for identifying an inhibitor of the activity of a membrane derived caspase, that includes, contacting a membrane fraction with a caspase substrate in the presence and absence of at least one candidate inhibitor; and comparing the levels of caspase substrate turnover, and therefrom identifying an inhibitor of the activity of a membrane derived caspase.
In yet another aspect, the present invention provides a method for identifying an enhancer of the activity of a membrane derived caspase, that includes, contacting a membrane fraction with a caspase substrate in the presence and absence of at least one candidate enhancer; and comparing the levels of caspase substrate turnover, and therefrom identifying an enhancer of the activity of a membrane derived caspase.
A further aspect of the present invention is a method for identifying an inhibitor or enhancer of the evolution of caspase processing within a membrane fraction, that includes, contacting a membrane fraction with at least one candidate inhibitor or candidate enhancer; and detecting the presence of large and small caspase subunits, and therefrom determining the level of caspase processing, wherein a decrease in processing indicates the presence of a caspase processing inhibitor, and wherein an increase in processing indicates the presence of a caspase processing enhancer.
In other embodiments, the present invention provides a method of identifying a compound that modulates membrane fraction derived caspase activity, that includes, incubating a membrane fraction, an inhibitor of apoptosis, and a caspase substrate in the presence and absence of at least one candidate compound under conditions and for a time sufficient to allow for the evolution of caspase activity; and comparing the levels of caspase substrate turnover, thereby identifying a compound that modulates membrane derived caspase activity.
In other embodiments, inhibitors and enhancers of the activity of a membrane derived caspase that are identified by the various methods are provided.
In the various embodiments, caspase activity is detected by measuring substrate turnover or caspase processing. In other embodiments, substrate turnover is measured by time course or endpoint analysis. In further embodiments, the membrane fraction comprises heavy or nuclear membranes. In yet further embodiments, the membrane fraction is derived from cells expressing an anti-apoptotic polypeptide. In even further embodiments, the membrane fraction is derived from non-apoptotic cells.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, the various references set forth below that describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.